Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated by reference herein as though set forth in full.
Gene expression measurements and analytical methods rest upon the assumption that a given messenger RNA sample provides a faithful representation of in vivo transcript levels at the time of extraction. In an ideal scenario, fully intact messenger RNA is reverse transcribed to high-quality cDNA for use in gene expression analysis studies, generating reliable and robust data. However, as a labile molecule, the integrity of RNA can be jeopardized at several points prior to, during, and post-extraction, adversely affecting the fidelity of gene expression measurements and hindering data interpretation and discovery. Accurately assessing RNA integrity prior to gene expression analysis on platforms such as microarrays and real-time quantitative PCR proves to be a critical step, requiring a highly sensitive and standardized RNA quality control method [1].
The current industry-standard technique for measuring RNA quality is microcapillary electrophoretic RNA separation, predominantly performed on the Agilent 2100 Bioanalyzer [2, 3]. The ‘lab-on-a-chip’ microfluidics technology and data visualization software offers multiple ways to visualize and evaluate RNA integrity, yet these broad-spectrum systems often lack sensitivity on the scale necessitated by RNA samples destined for gene expression analysis. While Bioanalyzer measurements provide a gross analytical assessment of RNA integrity, the proprietary RNA Integrity Number (RIN) scoring algorithm and visualization software has intrinsic limitations preventing in-depth RNA integrity profiles and cannot adequately predict the functional performance of RNA samples intended for gene expression analysis.
Clearly a need exists in the art for improved methods for assessing RNA integrity on a large scale.